U.S. patent application number 10/220401 was filed with the patent office on 2003-09-18 for quantifying target molecules contained in a liquid.
Invention is credited to Grassl, Bjorn, Hassmann, Jorg, Schulein, Jurgen.
Application Number | 20030175737 10/220401 |
Document ID | / |
Family ID | 7632936 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175737 |
Kind Code |
A1 |
Schulein, Jurgen ; et
al. |
September 18, 2003 |
Quantifying target molecules contained in a liquid
Abstract
The invention relates to a method for detecting and/or
quantifying first biopolymers contained in a liquid involving the
following steps: a) providing an electrode with a surface which is
made of plastic and which is coated with second biopolymers that
have a specific affinity to the first biopoymers to be detected; b)
bringing the electrode into contact with the liquid; c) applying a
predetermined voltage protocol to the electrode in order to effect
a concentration of the first biopolymers on the second biopolymers;
d) adding osmium tetroxide and bipyridine to the liquid, and; e)
measuring the redox signal coming off the electrode.
Inventors: |
Schulein, Jurgen; (Spardorf,
DE) ; Grassl, Bjorn; (Nurnberg, DE) ;
Hassmann, Jorg; (Erlangen, DE) |
Correspondence
Address: |
Mark S Ellinger
Fish & Richardson
Suite 3300
60 South Sixth Street
Minneapolis
MN
55402
US
|
Family ID: |
7632936 |
Appl. No.: |
10/220401 |
Filed: |
August 30, 2002 |
PCT Filed: |
February 28, 2001 |
PCT NO: |
PCT/DE01/00736 |
Current U.S.
Class: |
435/6.11 ;
205/777.5 |
Current CPC
Class: |
C12Q 1/6825 20130101;
C12Q 1/004 20130101; G01N 33/5438 20130101 |
Class at
Publication: |
435/6 ;
205/777.5 |
International
Class: |
C12Q 001/68; G01N
027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 1, 2000 |
DE |
100 09 715.4 |
Claims
1. A process for the detection and/or quantification of first
biopolymers present in a liquid, having the following steps: a)
provision of an electrode having a surface made of plastic which is
coated with second biopolymers which have a specific affinity to
the first biopolymers to be detected, b) bringing of the electrode
into contact with the liquid, c) application of a pre-specified
voltage program to the electrode, causing enrichment of the first
biopolymers at the second biopolymers, d) addition of osmium
tetroxide and bipyridine to the liquid, e) measurement of the redox
signal falling off at the electrode.
2. A process as claimed in claim 1, in which the plastic is an
electrically conductive composite material.
3. A process as claimed in one of the preceding claims, in which
the electrode consists entirely of plastic.
4. A process as claimed in one of the preceding claims, in which
the second biopolymers are bonded to a matrix, preferably made of
dextran or polyethylene glycol, applied to the surface of the
electrode.
5. A process as claimed in one of the preceding claims, in which
one of the following measurements is carried out in step e:
direct-voltage measurement, cyclovoltammetric measurement,
chronoamperometric measurement, chronovoltammetric measurement.
6. A process as claimed one of claims 1 to 3, in which a
differential pulse voltammogram is recorded in step e.
7. A process as claimed in one of claims 1 to 3, in which an
impendance spectrum is recorded in step e.
8. A process as claimed in one of claims 1 to 3, in which an
alternating-current signal is measured phase-sensitively in step
e.
9. A process as claimed in claim 5, in which the
alternating-current signal is superimposed on a direct-voltage
signal.
10. A process as claimed in one of the preceding claims, in which,
in order to quantify the first biopolymers, integration is carried
out via a peak of a measurement signal.
11. A process as claimed in one of the preceding claims, in which,
for multiple measurement, the electrode is rinsed or heated after
step e.
12. A process as claimed in one of the preceding claims, in which
the first biopolymers are subjected to a polymerase chain reaction
before step a.
Description
[0001] The invention relates to a process for the detection and/or
quantification of target molecules present in a liquid.
[0002] In accordance with the prior art, WO 96/01836 discloses a
chip for the detection of polynucleotide sequences. A multiplicity
of miniaturized reaction fields is provided on the chip, which is
made from a silicon substrate. A probe is connected to each of the
reaction fields. On immersion of the chip into a solution
containing the polynucleotide sequence to be detected,
hybridization with one of the probes provided occurs. The
hybridization can be detected, for example, by fluorophoric
labeling provided on the probe.
[0003] DE 198 08 884.1 describes a process for the detection of
chemical substances using two interacting fluorophoric groups which
are bonded to a molecule. In the case of specific adduction of the
molecule onto the chemical substance to be detected, the
interaction between the fluorophoric groups is modified.
[0004] WO 99/47700 relates to a process for the detection of a
target molecule by means of fluorescence. In this process, a probe
provided with a fluorophoric group is bound to a solid phase. In
the presence of the target sequence in the solution, a second
fluorophoric group is bound in the vicinity of the first
fluorophoric groups in such a way that radiation-free energy
transfer between the two fluorophoric groups can occur.
[0005] U.S. Pat. Nos. 5,312,572 and 5,871,918 describe processes
for the electrochemical detection of polynucleotide sequences. In
these processes, redox-active molecules which, on hybridization of
the polynucleotide sequence, bind to the double-stranded molecule
formed are added to the solution. The presence of a double-stranded
molecule of this type causes a measurable redox signal.
[0006] U.S. Pat. No. 5,591,578 describes a process for the
detection of polynucleotide sequences using redox indicators. In
this process, a probe which is complementary to the target
polynucleotide sequence is covalently bonded to an electrode.
Redox-active transition-metal complexes are covalently bonded to
the probe. On hybridization of the target polynucleotide sequence
with the probe, a redox signal can be measured at the
electrode.
[0007] DE 196 28 171 discloses a process for the purification and
enrichment of charge-carrying first molecules which have a specific
affinity to second molecules bonded to an electrode. When a
solution containing the first molecules is brought into contact
with the electrode, a voltage program is run through in such a way
that the first molecules are enriched at the electrode.
[0008] E. Palecek, Bioelectrochemistry and Bioenergetics 1985, 15,
275-295, discloses the use of osmium tetroxide compounds as
redox-active substance for the detection of double-stranded
biopolymers.
[0009] The processes disclosed in the prior art are time-consuming,
inconvenient or require complex equipment.
[0010] The object of the invention is to overcome the disadvantages
of the prior art. In particular, the aim is to indicate a
sensitive, simple and inexpensive electrochemical process for the
detection and/or quantification of small amounts of first
biopolymers present in a liquid.
[0011] This object is achieved by the features of claim 1.
Advantageous embodiments arise from the features of claims
2-12.
[0012] In accordance with the invention, provision is made for a
process for the detection and/or quantification of first
biopolymers present in a liquid, having the following steps:
[0013] a) provision of an electrode having a surface made of
plastic which is coated with second biopolymers which have a
specific affinity to the first biopolymers to be detected,
[0014] b) bringing of the electrode into contact with the
liquid,
[0015] c) application of a pre-specified voltage program to the
electrode, causing enrichment of the first biopolymers at the
second biopolymers,
[0016] d) addition of osmium tetroxide and bipyridine to the
liquid,
[0017] e) measurement of the redox signal falling off at the
electrode.
[0018] The proposed process enables sensitive detection of first
biopolymers present in a liquid. The use of electrodes provided
with a plastic surface enables the process to be carried out
inexpensively. In particular, the process also enables
quantification of the first biopolymers present in the liquid.
[0019] The term first and second biopolymers here is taken to mean,
in particular, proteins, peptides, DNA, RNA and the like. The first
biopolymer may be, in particular, a single-stranded DNA or RNA
which is complementary to the second biopolymer.
[0020] The second biopolymers are preferably covalently bonded to
the plastic surface. In combination with the proposed use of osmium
tetroxide and bipyridine, particularly high sensitivity is
achieved.
[0021] According to an advantageous embodiment, the plastic is an
electrically conductive composite material, for example a composite
of carbon fibers and polycarbonate. The electrode advantageously
consists entirely of the plastic. Such electrodes can be produced
in an inexpensive pressing process.
[0022] It is furthermore possible for the second biopolymers to be
bonded to a matrix, preferably made of dextran or polyethylene
glycol, applied to the surface of the electrode. The use of a
matrix of this type enables the coverage density of the surface of
the electrode with second biopolymers to be increased.
[0023] According to a further embodiment, one of the following
measurements is carried out in step e: direct-voltage measurement,
cyclovoltammetric measurement, chronoamperometric measurement,
chronovoltammetric measurement. Furthermore, a differential pulse
voltammogram or an impendance spectrum can be recorded in step e.
It is also possible to measure an alternating-current signal
phase-sensitively in step e. A direct-voltage signal may be
superimposed on the alternating-current signal. In order to
quantify the first biopolymers, integration can be carried out via
a peak of the measurement signal. The quantification parameter that
can be utilized is the separation between peak height and
background.
[0024] In order to carry out multiple measurements, the electrode
can be rinsed or heated after step e. Heating of the electrode
facilitates thermal denaturing of the first biopolymers. Certain
first biopolymers preferentially bind at a pre-specified
temperature. Heating or setting of the temperature enables the
specificity of the process to be increased further. The specificity
or stringency can also be increased by suitable setting of the pH
in the liquid.
[0025] The first biopolymers are advantageously subjected to a
polymerase chain reaction before step a. This enables the detection
of particularly small amounts of first biopolymers.
[0026] The process is explained in greater detail with reference to
the drawing and a working example.
[0027] The single FIGURE shows a differential pulse voltammogram of
an uncoated working electrode, a working electrode coated with
single-stranded oligonucleotides and a working electrode coated
with a hybridized oligonucleotide, in each case after treatment
with osmium tetroxide and bipyridine. The working electrodes each
consist of carbon composite material, which is preferably composed
of 30% of carbon fibers and 70% of polycarbonate. Oligonucleotides
containing the sequence 5'-GCC TTC CCA ACC ATT CCC TTA-3' were
covalently bonded to the surface of the working electrodes using
carbodiimide by a standard method. The coverage density was 15
fmol/mm.sup.2. The hybridization of the oligonucleotides was
carried out in a buffered solution of 0.5-fold TBE (TRIS borate
EDTA) 0.5 M NaCl and 100 fmol/.mu.l of complementary
oligonucleotides. After the hybridization, the working electrodes
were washed stringently.
[0028] An untreated working electrode, a working electrode coated
with single-stranded oligonucleotides and a working electrode
coated with hybridized oligonucleotides were subsequently each
dipped in a solution of 2 mM OSO.sub.4 and 13 mM bipyridine for 30
seconds. The measurement was carried out with the aid of a platinum
counterelectrode and an Ag/AgCl reference electrode using an
Ecochemie PGSTAT 10 Autolab.
[0029] The hybridization of the target oligonucleotides at the
working electrode can be accelerated by application of a voltage.
The coverage density of oligonucleotides on the surface of the
working electrode can be increased by addition of salt during the
coating or by basic pretreatment of the surface. For example, a
coverage density of 85 fmol/mm.sup.2 can be achieved in a 10 mM
MgCl.sub.2 solution. In the case of pretreatment of the surface for
three hours in 5 M NaOH, a coverage density of 750 fmol/mm.sup.2
can be achieved.
[0030] If a plurality of measurements are to be carried out one
after the other, an opposite voltage can be applied to the
electrode after step d. In addition, the electrode can be rinsed
and/or heated after step e. The heating of the electrode
facilitates thermal denaturing of the first biopolymers. However,
heating or setting of the temperature of the electrode also enables
the specificity of the process to be increased since prespecified
first biopolymers bind at a specific temperature.
* * * * *